摘要
目的 评价全基因组测序技术在耐药肺结核诊断中的应用价值,为耐药肺结核的早期诊断提供参考.方法 以2019年1月1日至2021年12月31日深圳市宝安区结核病定点医院确诊登记肺结核患者,痰培养阳性结核分枝杆菌菌株的8种抗结核药物表型药敏试验结果为金标准,将菌株全基因组测序的结果上传至SAM-TB结核分枝杆菌全基因组测序平台获得各菌株基因突变和耐药情况,比较各菌株基因组耐药的灵敏度、特异度、符合率和Kappa值,评价全基因组测序技术在耐药肺结核诊断中的应用价值.结果 本次研究共纳入1327例培养阳性菌株,全基因组测序总体耐药率为20.35％(270/1327).86.30％(233/270)的耐药菌株至少对异烟肼、利福平、乙胺丁醇或链霉素等4种一线抗结核药物中的1种耐药,一、二线抗结核药物耐药率最高的分别为链霉素(10.63％)和氧氟沙星(3.01％).耐药菌株中,对异烟肼耐药的菌株主要发生了katG、fabG1、inhA和ahpC 4种基因突变,对链霉素和对氨基水杨酸耐药的菌株均产生了 3种基因突变,对乙胺丁醇、卷曲霉素和氧氟沙星耐药的菌株均产生了 2种基因突变,对利福平、卡那霉素耐药的菌株则各产生了 1种基因突变.全基因组测序技术预测异烟肼、利福平耐药性的灵敏度、特异度均分别在93.44％(95％CI 84.05～98.18)、97.55％(95％CI 96.54～98.33),对乙胺丁醇、链霉素耐药性预测的灵敏度分别为80.77％(95％CI 60.65～93.45)、70.18％(95％CI 62.72～70.92),特异度分别为99.08％(95％CI 98.39～99.52)、98.18％(95％CI 97.24～98.87);对4种二线抗结核药物耐药性预测的灵敏度在50％(95％CI 6.76～93.24)至 100％(95％CI 88.42～100.00)之间,特异度均在98.86％(95％CI 98.13～99.36)及以上.结论 全基因组测序技术对异烟肼、利福平和氧氟沙星等耐药肺结核诊断的灵敏度、特异度均较高,具有较高的临床应用价值.但对于卷曲霉素、链霉素、对氨基水杨酸等耐药肺结核的诊断价值仍需更多的研究加以证实.
Abstract
Objective To evaluate the application value of whole-genome sequencing technology to diagnostic the drug-resistant tuberculosis,and provide reference for the early diagnosis of drug-resistant tuberculosis.Method The phenotypic susceptibility results of 8 kinds of anti-tuberculosis drugs with sputum culture positive strains were regard as gold standard,which patients were confirmed and registered in the designated medical institution of Bao'an District from January 1,2019 to December 31,2021.And the strains were inactivated before DNA was extracted for whole template sequence.The sequence results were uploaded to the platform to obtain the mutation sites of each strain.With reference to the drug resistance gene loci reported in previous literature,the sequence results were compared with the phenotypic drug susceptibility results,and the sensitivity,specificity,coincidence rate and Kappa value were used to evaluate the application value of whole-genome sequencing technology.Result The total drug resistance rate of 1327 culture-positive strains was 20.35％(270/1327).86.30％(233/270)of the resistant strains were resistant to at least one of the four first-line anti-tuberculosis drugs,including isoniazid,rifampicin,ethambutol and streptomycin.The highest resistance rates of first and second-line anti-tuberculosis drugs were streptomycin(10.63％)and ofloxacin(3.01％).katG,fabGl,inhA and ahpC mutated genes were found in isoniazid-resistant strains.Three genetic mutations were found in resistant strains to streptomycin and para-aminosailcylic acid.Two genetic mutations were found in resistant strains to ethambutol,capreomycin and ofloxacin.There were only one genetic mutation in resistant strains to rifampicin and kanamycin.The sensitivity and specificity of whole-genome sequencing for predicting resistance to isoniazid and rifampicin were above 93.44％(95％CI 84.05-98.18),97.55％(95％CI 96.54-98.33),respectively.The sensitivity and specificity of the whole-genome sequencing technology for predicting resistance to ethambutol and streptomycin was 80.77％(95％CI 60.65-93.45)and 70.18％(95％CI 62.72-70.92),99.08％(95％CI 98.39-99.52)and 98.18％(95％CI 97.24-98.87),respectively.For the four second-line anti-tuberculosis drugs,the sensitivity of the whole-genome sequencing technology was from 50％(95％CI 6.76-93.24)to 100％(95％CI 88.42-100.00),the specificity was 98.86％(95％CI 98.13-99.36)or above.Conclusion The whole-genome sequencing technology has high application value in the diagnosis of drug-resistant tuberculosis for isoniazid,rifampicin,and ofloxacin,with the higher sensitivity and specificity.But more studies are needed to confirm it's diagnosis value to drug-resistant pulmonary tuberculosis for capreomycin,streptomycinand para-aminosailcylic acid.
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